JP2023519164A - Apparatus and method for sample analysis - Google Patents

Abstract

The present disclosure relates generally to devices and methods for performing epitachophoresis. Epitacophoresis can be used, for example, to perform sample analysis such as by selective separation, detection, extraction and/or preconcentration of target analytes such as DNA, RNA and/or other biomolecules. The target analytes can be collected after epitacophoresis and used for desired downstream applications and further analysis.

Description

FIELD OF THE DISCLOSURE The present disclosure relates generally to the field of electrophoresis, and more particularly to the selective separation, detection, extraction, isolation and purification of samples, such as those containing nucleic acids, e.g., by devices and methods for epitakophoresis. and/or sample analysis by (pre)concentration.

For example, the use of nucleic acid-based analytical techniques such as DNA sequencing, RNA sequencing, and gene expression profiling are not only commonplace in a variety of research applications, but are rapidly becoming part of many therapeutic regimens. . For example, determination of gene expression levels in tissues is of great importance for accurately diagnosing human disease and is increasingly used to determine the course of patient treatment. In some cases, pharmacogenomic methods can identify patients who are more likely to respond to a particular drug, leading to new therapeutic approaches.

One important source of this type of information is in the form of preserved biological specimens, eg tissues that have undergone a fixation process. In some examples, such fixation processes include chemical fixation by using cross-linking fixatives, such as aldehyde-based fixatives. For example, formalin-based solutions can often be used to prepare formalin-fixed, paraffin-embedded tissue (“FFPET”) samples. FFPET samples are routinely made from biopsy specimens taken from patients undergoing diagnosis and/or treatment regimens for a variety of different diseases. These samples are usually associated with corresponding clinical records and often play an important role in diagnosing and determining treatment modalities. For example, tumor biopsy FFPET samples are often correlated with cancer staging, patient survival, and treatment regimens, thereby providing potentially rich information that can be cross-referenced and correlated with, for example, gene expression patterns. offer. However, the poor quality and quantity of nucleic acids isolated from FFPET samples using current methodologies has led to their general underutilization. For example, RNA isolated from FFPET samples is often moderately to highly degraded and fragmented, a significant drawback to its use in diagnostic and/or therapeutic applications. In addition to being degraded and fragmented, chemical modification of RNA by formalin can limit the binding of oligo-dT primers to the polyadenylate tails of RNA, impeding the efficiency of processes involving the use of reverse transcription. there is a possibility.

Furthermore, conventional techniques for the extraction, isolation and/or purification of nucleic acids from stored samples, such as chemically fixed samples that have been subjected to cross-linking fixatives, e.g., aldehyde-based fixatives, often involve: It is time consuming, results in poor product quality and/or involves the use of harsh chemicals such as organic solvents such as xylene. Additionally, conventional techniques typically use column and/or bead-based processes that are costly, labor intensive, and not well suited for automation. Accordingly, there is a need for further development of devices and methods for analyzing samples such as FFPET samples, which are stored.

The present disclosure generally relates to methods of isolating and/or purifying one or more nucleic acids from a sample comprising one or more nucleic acids, optionally wherein the sample comprises a chemically fixed sample, further optionally formalin Comprising a fixed paraffin-embedded tissue (“FFPET”) sample, the method comprises: a. providing a device for performing epitakophoresis (“ETP”); b. providing a sample comprising said one or more nucleic acids; c. performing one or more epitakophoresis runs by performing ETP using the device to focus the one or more nucleic acids into one or more focusing zones; d. collecting said one or more nucleic acids by collecting said one or more focused zones containing said one or more nucleic acids; thereby obtaining one or more isolated and/or purified nucleic acids; and the nucleic acid may comprise DNA and/or RNA. In some embodiments, prior to step c, a sample solution may be prepared from the sample containing one or more nucleic acids. In some embodiments, the sample may include any one or more of an FFPET sample, an FFPET curl sample, and/or a sample solution prepared from any of the samples. In some embodiments, the sample may comprise an FFPET sample, and the FFPET sample solution is prepared from the FFPET sample. In some embodiments, nucleic acids can include DNA and/or RNA. In some embodiments, isolated and/or purified nucleic acids may comprise DNA and/or RNA. In some embodiments, isolated and/or purified nucleic acid collected from any single ETP run may contain both DNA and RNA, optionally DNA and RNA are isolated/purified simultaneously, Collected. In some embodiments, isolated and/or purified nucleic acid substantially or completely collected from any single ETP run can comprise either DNA or RNA, i.e., DNA and RNA are , substantially or completely isolated/purified separately (ie, not simultaneously) and collected. In some embodiments, isolated and/or purified nucleic acid collected from any single ETP run can comprise a mixture of DNA and RNA, wherein the collected mixture of DNA and RNA comprises one or more DNA and/or RNA are subjected to downstream separations prior to use in IVD assays. In some embodiments, the amount of isolated and/or purified nucleic acid, when measured by a fluorometer-based method, is comparable to the amount of nucleic acid obtained using column-based or bead-based protocols. can be relatively large. In some embodiments, the quality of the isolated and/or purified nucleic acids, as measured by quality control qPCR-based methods, is higher than that of nucleic acids obtained using column-based protocols or bead-based protocols. Can be expensive compared to quality. In some embodiments, 1.25-fold or greater, 1.5-fold or greater, 1.75-fold or greater, 2.0-fold compared to the amount of nucleic acid obtained using column-based or bead-based protocols. Collect more than 2.25 times, 2.5 times more, 2.75 times more, 3 times more, 4 times more, 5 times more, 10 times more, 100 times more, or 1000 times more nucleic acids be able to. In some embodiments, the method removes 1% or less, 1% or more, 5% or more, 10% or more of the one or more nucleic acids contained in the original sample to be isolated and/or purified, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 85% Greater than 90%, greater than 95%, or greater than 99% can be provided. In some embodiments, the method includes one or more isolated and/or purified 1% or less, 1% or more, 5% or more, 10% or more, 15% or more, 25% or more, 20% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more of the nucleic acid, Purities of 55% or greater, 60% or greater, 65% or greater, 70% or greater, 85% or greater, 90% or greater, 95% or greater, or 99% or greater can be provided. In some embodiments, the quality of the isolated and/or purified nucleic acids can be determined by quality control qPCR. In some embodiments, isolated and/or purified nucleic acids can be of any desired size. In some embodiments, the isolated and/or purified nucleic acid is 5 nt or less, 10 nt or less, 20 nt or less, 30 nt or less, 50 nt or less, 100 nt or less, 1000 nt or less, 10,000 nt or less, 100,000 nt or less, 1 ,000,000nt or less, or 1,000,000nt or more in size. In some embodiments, the method may include one or more pretreatment steps. In some embodiments, the pretreatment step can include lysing the chemically fixed sample and/or preparing a sample solution. In some embodiments, the chemically fixed sample may comprise FFPET curls and the sample solution comprises FFPET sample solution prepared by dissolving the FFPET curls. In some embodiments, trailing electrolyte (“TE”) buffer can be added after dissolution of the FFPET curl.

Additionally, the present disclosure generally relates to methods for isolating and/or purifying one or more nucleic acids from formalin-fixed, paraffin-embedded tissue (“FFPET”) samples, which methods perform epitachophoresis (ETP). providing a device for; b. providing an FFPET sample containing nucleic acids; c. preparing an FFPET sample solution from said FFPET sample; d. performing one or more epitakophoresis runs by performing ETP using the device to focus the one or more nucleic acids into one or more focus zones; and e. collecting said one or more nucleic acids by collecting said one or more focused zones containing said one or more nucleic acids; thereby producing one or more isolated and/or purified nucleic acids; including obtaining

Additionally, the present disclosure generally relates to methods of isolating and/or purifying one or more nucleic acids from a formalin-fixed, paraffin-embedded tissue (“FFPET”) sample, the nucleic acids including DNA and RNA, the methods comprising: providing a device for performing epitakophoresis (ETP); b. providing an FFPET sample comprising nucleic acids, optionally comprising one or more FFPET curls; c. preparing an FFPET sample solution from the FFPET sample, optionally prepared by dissolving one or more FFPET curls and adding a trailing electrolyte (“TE”) buffer; preparing; d. performing one or more epitakophoresis runs by performing ETP using the device to focus the one or more nucleic acids into one or more focusing zones; and e. collecting said one or more nucleic acids by collecting said one or more focused zones containing said one or more nucleic acids; thereby obtaining one or more isolated and/or purified nucleic acids; including.

Additionally, the present disclosure generally relates to methods of isolating and/or purifying one or more nucleic acids from a formalin-fixed, paraffin-embedded tissue (“FFPET”) sample, the nucleic acids including DNA and RNA, the methods comprising: providing a device for performing epitakophoresis (ETP); b. providing an FFPET sample comprising nucleic acids, optionally comprising one or more FFPET curls; c. preparing an FFPET sample solution from the FFPET sample, optionally prepared by dissolving one or more FFPET curls and adding a trailing electrolyte (“TE”) buffer; preparing; d. performing one or more epitakophoresis runs by performing ETP using the device to focus the one or more nucleic acids into one or more focusing zones; and e. collecting said one or more nucleic acids by collecting said one or more focused zones containing said one or more nucleic acids; thereby obtaining one or more isolated and/or purified nucleic acids; and further, isolated and/or purified nucleic acid collected from any single ETP run includes both DNA and RNA.

Additionally, the present disclosure generally relates to methods of isolating and/or purifying one or more nucleic acids from a formalin-fixed, paraffin-embedded tissue (“FFPET”) sample, the nucleic acids including DNA and RNA, the methods comprising: providing a device for performing epitakophoresis (ETP); b. providing an FFPET sample comprising nucleic acids, optionally comprising one or more FFPET curls; c. preparing an FFPET sample solution from the FFPET sample, optionally prepared by dissolving one or more FFPET curls and adding a trailing electrolyte (“TE”) buffer; preparing; d. performing one or more epitakophoresis runs by performing ETP using the device to focus the one or more nucleic acids into one or more focusing zones; and e. collecting said one or more nucleic acids by collecting said one or more focused zones containing said one or more nucleic acids; further comprising isolating and collecting said one or more nucleic acids collected from any single ETP run; /or a purified nucleic acid substantially or completely comprises either DNA or RNA, i.e., DNA and RNA are substantially or completely isolated/purified separately (i.e., not simultaneously). , is collected.

Additionally, the present disclosure generally relates to methods of isolating and/or purifying one or more nucleic acids from a formalin-fixed, paraffin-embedded tissue (“FFPET”) sample, the nucleic acids including DNA and RNA, the methods comprising: providing a device for performing epitakophoresis (ETP); b. providing an FFPET sample comprising nucleic acids, optionally comprising one or more FFPET curls; c. preparing an FFPET sample solution from the FFPET sample, optionally prepared by dissolving one or more FFPET curls and adding a trailing electrolyte (“TE”) buffer; preparing; d. performing one or more epitakophoresis runs by performing ETP using the device to focus the one or more nucleic acids into one or more focusing zones; and e. collecting said one or more nucleic acids by collecting said one or more focused zones containing said one or more nucleic acids; isolating and/or collected from any single ETP run; The purified nucleic acid comprises a mixture of DNA and RNA, and the collected DNA and RNA mixture is subjected to downstream separation prior to using the DNA and/or RNA in one or more IVD assays. In some embodiments, the isolated and/or purified nucleic acid is subjected to one or more in vitro diagnostic (“IVD”) assays.

1 provides a schematic diagram of an exemplary apparatus for performing epitachophoresis. FIG. 1 provides a top view schematic of an exemplary apparatus for performing epitachophoresis. In FIG. 2A, numbers 1-7 refer to: 1. outer circular electrode;2. Termination electrolyte reservoir;3. 4. a leading electrolyte, optionally contained within the gel or hydrodynamically separated from the terminating electrolyte; 5. Leading electrolyte electrode/collection reservoir; central electrode;6. and7. Boundary between leading and terminating electrolytes with sample ions focused in between; the symbols r and d are used to represent the leading electrolyte reservoir radius and distance moved by the LE/TE boundary, respectively . FIG. 1 provides a side-view schematic diagram of an exemplary apparatus for performing epitakophoresis. In FIG. 2B, the numbers 1-8 refer to:1. outer circular electrode;2. Termination electrolyte reservoir;3. 4. a leading electrolyte, optionally contained within the gel or hydrodynamically separated from the terminating electrolyte; 5. Leading electrolyte electrode/collection reservoir; 6. center electrode; and7. 8. a boundary between the leading and terminating electrolytes with sample ions focused therebetween; Bottom support; the symbols r and d are used to represent the radius and distance, respectively, of the leading electrolyte reservoir moved by the LE/TE boundary. 1 provides a schematic diagram of an exemplary apparatus for performing epitachophoresis. 1 provides a schematic diagram of an exemplary apparatus for performing epitachophoresis. In FIG. 4, the numbers 1-10 refer to:1. outer circular electrode;2. Termination electrolyte reservoir;3. 4. a leading electrolyte, optionally contained within the gel or hydrodynamically separated from the terminating electrolyte; 4. openings to pre-electrolyte/collection reservoirs; 6. center electrode; and7. 8. the boundary between the leading and terminating electrolytes with sample ions focused therebetween; 9. bottom support; 10. tubing connection to the preceding electrolyte reservoir; Leading electrolyte reservoir. FIG. 1 provides a schematic diagram of an exemplary apparatus for performing epitakophoresis, in which the sample is loaded between the leading electrolyte loading and the terminating electrolyte loading. 1 provides a schematic diagram of an apparatus for performing epitachophoresis and refers to the equations set forth in Example 2. FIG. FIG. 6B provides a graph depicting relative velocity in cm versus distance d traveled in cm when the exemplary apparatus for epitachophoresis ( FIG. 6A ) is operated using a constant current. In the example shown in FIG. 6B, a radius value of 5 and a starting velocity value of 1 were used. FIG. 6B provides a graph depicting relative velocity at distance d versus distance d traveled in cm when the exemplary apparatus for epitachophoresis (FIG. 6A) is operated using constant voltage. In the example shown in FIG. 6C, a radius value of 5 and a starting velocity value of 1 were used. FIG. 6B provides a graph depicting relative velocity at distance d versus travel distance d in cm when the exemplary apparatus for epitachophoresis ( FIG. 6A ) is operated using constant power. In the example shown in FIG. 6D, a radius value of 5 and a starting velocity value of 1 were used. 2 provides an image of an epitakophoresis device used to concentrate samples according to Example 3. FIG. 4 provides an image of an exemplary apparatus for epitachophoresis used according to Example 4; 4 provides an image of an exemplary apparatus for epitachophoresis used to focus a sample into a focus zone according to Example 4. FIG. 4 provides an image of an exemplary apparatus for epitachophoresis used to focus a sample into a focus zone according to Example 4. FIG. 5 provides an image of an exemplary device for epitachophoresis used according to Example 5. FIG. 5 provides a schematic diagram of an exemplary apparatus for epitachophoresis used according to Example 5. FIG. In FIG. 9B, the reference numbers refer to dimensions in millimeters. 5 provides an image of an exemplary apparatus for epitachophoresis used to focus a sample into a focus zone according to Example 5. FIG. 5 provides an image of an exemplary apparatus for epitachophoresis used to focus a sample into a focus zone according to Example 5. FIG. 5 provides an image of an exemplary apparatus for epitachophoresis used to focus a sample into a focus zone according to Example 5. FIG. 5 provides images of an exemplary apparatus for epitakophoresis used to separate and focus two different samples into a focus zone according to Example 5. FIG. 10 provides images of an exemplary apparatus for epitakophoresis according to Example 6. FIG. 7 provides images of an exemplary epitakophoresis device according to Example 7. FIG. 5 provides a schematic diagram of an exemplary epitakophoresis device according to Example 7. FIG. "a" corresponds to the central collection well and "b" corresponds to the leading electrolyte reservoir. 8 provides an image of an exemplary conductivity measurement probe for use in an epitakophoresis device according to Example 7; 14B provides an image showing a magnified view of the conductivity measuring probe shown in FIG. 14A. 5 provides an image of an exemplary epitacophoresis device having a conductivity probe according to Example 7; 6 provides conductivity traces for an exemplary epitakophoresis device run according to Example 7. FIG. 8 provides an image of an exemplary epitakophoresis device having a conductivity sensing probe positioned below a semi-permeable membrane according to Example 7; FIG. 5 provides an image of an exemplary bottom substrate incorporating two conductivity sensing probes connected via dedicated channels in the central post. 2 provides an image of an exemplary epitakophoresis apparatus showing focusing of a fluorescein-labeled DNA ladder sample according to Example 7; 10 provides traces showing resistivity changes of LE/TE transitions monitored by a surface conductive cell for an exemplary epitakophoresis device run according to Example 7; 2 provides absorbance spectra of collected fractions of original and DNA ladder samples before and after performing epitakophoresis according to Example 7. FIG. 7 provides electropherograms of DNA ladder samples before and after epitacophoresis run measured via Bioanalyzer separation according to Example 7. FIG. 10 provides voltage profiles of three independent ETP runs according to Example 8; 10 provides an image of the ETP device during ETP execution according to Example 9; 10 provides fluorescence-based images of an ETP device taken during an ETP run according to Example 9; 10 provides images of an ETP device taken during an ETP run according to Example 9; 10 provides electropherograms of analyzed focused and collected ETP samples according to Example 9. FIG. 10 provides images captured using an infrared-based thermal imaging camera during an ETP run according to Example 10; 10 provides fluorescence-based images captured during an ETP run according to Example 10; 10 presents data on voltage and temperature changes over time during ETP run according to Example 10; 11 provides images of the ETP experimental setup according to Example 11. FIG. 11 provides images of the ETP experimental setup according to Example 11. FIG. 10 provides images of an ETP run using Brilliant Blue dye according to Example 11 as a sample marker. 10 provides images of an ETP run using Brilliant Blue dye according to Example 11 as a sample marker. 2 provides data from ETP-based isolation/purification of dsDNA from FFPET samples according to Example 12. The amount of dsDNA obtained by ETP-based isolation/purification was compared to the amount obtained using the Promega column-based method described in Example 12. 12 provides data from ETP-based isolation/purification of RNA from FFPET samples according to Example 12. The amount of RNA obtained by ETP-based isolation and purification was compared to the amount obtained using the Promega column-based method described in Example 12. 4 presents data from size-based analysis of dsDNA isolated/purified by ETP-based isolation/purification compared to four different control samples according to Example 13. FIG. 13 presents data from a qPCR-based analysis of the quality of dsDNA isolated/purified by ETP-based isolation/purification compared to the quality of dsDNA obtained by the Promega column-based method according to Example 13. . 2 provides data from ETP-based isolation/purification of dsDNA from FFPET samples according to Example 14. The amount of dsDNA obtained by ETP-based isolation/purification was compared to that obtained using the KAPA bead-based method. 14 provides data from ETP-based isolation/purification of RNA from FFPET samples according to Example 14. The amount of RNA obtained by ETP-based isolation and purification was compared to that obtained using the KAPA bead-based method described in Example 14. 2 provides data for DNase I-based analysis of nucleic acids obtained by ETP-based isolation/purification of FFPET samples according to Example 15. 2 provides data on size-based analysis of nucleic acids obtained by ETP-based isolation/purification of FFPET samples before and after DNase I treatment according to Example 15. FIG. Data are provided for comparison of the state-of-the-art FFPET extraction kit described in Example 16 with the ETP method. FIG. 16 provides sequencing data for nucleic acids extracted in Example 16. FIG.

DEFINITIONS As used herein, the singular forms “a,” “an,” and “the” refer to plural referents unless the context clearly dictates otherwise. including. All technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, unless defined otherwise.

As used herein, the term "isokinetic electrophoresis" generally refers to the formation of boundaries or interfaces between materials (e.g., between charged particles and other materials in solution) using an electric field. It refers to the separation of charged particles by ITP generally employs multiple electrolytes, the electrophoretic mobility of the sample ions being less than that of the leading electrolyte (LE) placed in the apparatus for ITP, and the trailing electrolyte (TE). greater than the electrophoretic mobility of The leading electrolyte (LE) generally contains ions with relatively high mobility and the trailing electrolyte (TE) generally contains ions with relatively low mobility. The TE and LE ions are selected to have lower and higher effective mobilities, respectively, than the target analyte ions of interest. That is, the effective mobility of the analyte ions is higher than TE and lower than LE. These target analytes have the same sign of charge as the LE and TE ions (ie co-ions). The applied electric field pushes the LE ions away from the TE ions and pulls the TE ions back. Moving interfaces are formed between adjacent and successive TE and LE zones. This creates a region of electric field gradient (typically from low field for LE to high field for TE). Analyte ions in the TE overtake TE ions but cannot overtake LE ions and can accumulate at the interface between TE and LE (forming a "focus" or "zone of focus"). . Alternatively, target ions in the LE are overtaken by LE ions and also accumulate at the interface. With judicious choice of LE and TE chemistries, ITP is fairly universally applicable, can be achieved by first dissolving samples in either or both TE and LE electrolytes, and has very low electrical conductivity. A small amount of background electrolyte may not be required.

As used herein, the term "epitacophoresis" generally refers to circular or spheroidal and/or concentric devices, such as by use of circular/concentric and/or polygonal devices described herein. and/or refers to methods of electrophoretic separation performed using circular and/or concentric electrode configurations. Due to the circular/concentric or other polygonal arrangement used during epitacophoresis, the cross-sectional area changes during ion and zone migration, unlike conventional isotachophoresis devices, resulting in zone migration. is not constant in time due to cross-sectional area changes. Therefore, the epitakophoresis configuration does not strictly follow the conventional isotachophoresis principle, in which zones move at a constant velocity. Despite these significant differences shown here, epitakophoresis is a boundary between materials that can have different electrophoretic mobilities (e.g., between charged particles and other materials in solution). Or it can be used to efficiently separate and focus charged particles by using an electric field to form an interface. LE and TE can also be used for epitachophoresis, as described for use with ITP. In some embodiments, epitachophoresis can be performed using constant current, constant voltage, and/or constant power. In some embodiments, epitakophoresis can be performed using varying current, varying voltage, and/or varying power. In some embodiments, epitakophoresis can be described as generally circular or spherical in shape so that the basic principles of epitakophoresis can be achieved as described herein. It can be achieved within the context of possible device and/or electrode placement. In some embodiments, epitakophoresis can be generally described as polygonal in shape so that the basic principles of epitakophoresis can be achieved as described herein. It can be accomplished within the context of device and/or electrode placement. In some embodiments, epitachophoresis can be achieved by any non-linear sequential arrangement of electrodes, such as electrodes arranged in a circular shape and/or electrodes arranged in a polygonal shape. can.

As used herein, terms such as "in vitro diagnostic applications (IVD applications)," "in vitro diagnostic methods (IVD methods)," "in vitro diagnostic assays," and the like generally refer to a subject, optionally a human subject. Refers to any use and/or method and/or device capable of evaluating a sample for diagnostic and/or monitoring purposes, such as identification of diseases such as cancer. In some embodiments, the sample may comprise nucleic acids and/or target nucleic acids from a subject and/or sample, optionally further wherein the nucleic acids are stored samples, e.g., stored tissue samples, e.g. Derived from FFPET samples. In some embodiments, an epitakophoresis device can be used as an in vitro diagnostic device. In some embodiments, target analytes enriched/enriched/isolated/purified by epitakophoresis can be used in downstream in vitro diagnostic assays. In some embodiments, in vitro diagnostic assays may comprise nucleic acid sequencing, such as DNA sequencing, such as RNA sequencing. In some embodiments and IVD assays may include gene expression profiling. In some embodiments, in vitro diagnostic methods can be, but are not limited to, any one or more of the following: staining, immunohistochemical staining, flow cytometry, FACS, fluorescence activity droplet sorting, image analysis, hybridization, DASH, molecular beacons, primer extension, microarray, CISH, FISH, fiber FISH, quantitative FISH, flow FISH, comparative genomic hybridization, blotting, western blotting, southern blotting, eastern blotting , far-western blotting, southwestern blotting, northwestern blotting, and northern blotting, enzyme assays, ELISA, ligand binding assays, immunoprecipitation, ChIP, ChIP-seq, ChIP-ChiP, radioimmunoassay, fluorescence polarization, FRET, surface plasmon resonance , filter binding assay, affinity chromatography, immunocytochemistry, gene expression profiling, DNA profiling PCR, DNA microarray, serial analysis of gene expression, real-time polymerase chain reaction, differential display PCR, RNA-seq, mass spectrometry, DNA methylation detection , acoustic energy, lipidomic-based analysis, quantification of immune cells, detection of cancer-associated markers, affinity purification of specific cell types, DNA sequencing, next-generation sequencing, detection of cancer-associated fusion proteins, and chemotherapy resistance. Detection of sex-related markers.

As used herein, the terms "leading electrolyte" and "leading ion" are generally compared to those of the sample ion and/or trailing electrolyte of interest used during ITP and/or epitachophoresis. , refers to ions with higher effective electrophoretic mobilities. In some embodiments, the preceding electrolytes for use in cationic epitacophoresis include, but are not limited to, chloride buffered to the desired pH by a suitable base such as histidine, TRIS, creatinine, Sulfates and/or formates may be included. In some embodiments, pre-electrolytes for use in anionic epitacophoresis can include, but are not limited to, potassium, ammonium and/or sodium along with acetate or formate. In some embodiments, increasing the concentration of the leading electrolyte can result in a proportional increase in sample zone and a corresponding increase in current (power) for a given applied voltage. Typical concentrations can generally range from 10-20 mM. However, higher concentrations may be used.

As used herein, the terms “trailing electrolyte,” “trailing ion,” “terminating electrolyte,” and “terminating ion” are generally used during ITP and/or epitachophoresis for the purposes of sample ions and/or ions that have a lower effective electrophoretic mobility compared to the electrophoretic mobility of the preceding electrolyte. In some embodiments, trailing electrolytes for use in cationic epitacophoresis include, but are not limited to, MES, MOPS, acetate, glutamate, and other anions of weak acids and low mobility anions. can contain. In some embodiments, trailing electrolytes for use in anionic epitacophoresis include, but are not limited to, reactive hydroxonium ions at the migrating boundary formed by any weak acid during epitacophoresis. be able to.

As used herein, the term "focusing zone" generally refers to a volume of solution containing components that have been concentrated ("focused") as a result of performing epitakophoresis. A focused zone may be collected or removed from the device, and the focused zone may contain an enriched and/or concentrated amount of a desired sample, eg, target analyte, eg, target nucleic acid. In the epitakophoresis methods described herein, the target analyte is generally focused to the center of the device, eg, a circular or spheroidal or other polygonal device.

As used herein, the terms "band" and "ETP band" generally refer to a band that is associated with other ions, analytes, or samples during electrophoretic (e.g., isotachophoresis or epitacophoresis) migration. refers to a zone of separately migrating ions, analytes, or samples (eg, a focus zone). A focal zone within an epitakophoresis device may alternatively be referred to as an "ETP band." In some embodiments, an ETP band can contain one or more types of ions, analytes, and/or samples. In some instances, an ETP band can comprise a single type of analyte for which separation from other materials present in the sample is desired, such as separation of the target nucleic acid from cell debris. In some instances, an ETP band can contain more than one desired analyte, such as polypeptides or nucleic acid sequences that are very similar in sequence, such as allelic variants. In some examples, the ETP bands can contain analytes of similar size or different electrophoretic mobilities. In such cases, more than one desired analyte may be separated by further ETP runs, e.g. under different conditions that promote separation of said more than one analyte, and/or said one of analytes may be separated by other techniques known in the art for the separation of analytes, such as those described herein. In some embodiments, the ETP bands can be collected and optionally subjected to further analysis after one or more ETP-based isolation/purification and collection. In some embodiments, an ETP band can comprise one or more target analytes that have or have undergone ETP-based isolation/purification and optionally collection, eg, as part of an ETP run.

As used herein, the term "target nucleic acid" is intended to mean any nucleic acid to be detected, measured, amplified, isolated, purified, and/or further assayed and analyzed. . A target nucleic acid can comprise any single and/or double stranded nucleic acid. A target nucleic acid can exist as an isolated nucleic acid fragment or can be part of a larger nucleic acid fragment. Target nucleic acids can be from essentially any supply of cultured microorganisms, uncultured microorganisms, complex biological mixtures, samples containing biological samples, tissues, serum, old or preserved tissues or samples, environmental isolates, etc. It can be derived or isolated from a source. Furthermore, the target nucleic acid may be cDNA, RNA, genomic DNA, cloned genomic DNA, genomic DNA library, enzymatically fragmented DNA or RNA, chemically fragmented DNA or RNA, physically fragmented including or derived from DNA or RNA, etc. In some embodiments, the target nucleic acid can comprise the entire genome. In some embodiments, a target nucleic acid may comprise the total nucleic acid content of a sample and/or biological sample, eg, an FFPET sample. Targets can come in a variety of different forms, including, for example, simple or complex mixtures or substantially purified forms. For example, a target can be part of a sample that contains other components, or it can be the sole or major component of the sample. Also, the target nucleic acid can have either known or unknown sequence.

As used herein, the term "target microorganism" refers to any organism found in samples such as blood, plasma, other body fluids, biological samples, and/or tissue, such as those associated with an infectious condition or disease. It is intended to mean unicellular or multicellular microorganisms. Examples include bacteria, archaea, eukaryotes, viruses, yeast, fungi, protozoa, amoebas and/or parasites. Further, the term "microorganism" generally refers to a microorganism capable of causing disease, whether a disease or a disease-causing microorganism is being referred to.

As used herein, the term "biomarker" or "biomarker of interest" refers to tissue, blood, plasma, urine, and/or refers to biological molecules found in other bodily fluids. Biomarkers can be used to ascertain how well the body responds to treatment for a disease or condition. In the context of cancer, biomarkers refer to biological substances that indicate the presence of cancer in the body. A biomarker can be a molecule secreted by a tumor or a specific response of the body to the presence of cancer. Genetic, epigenetic, proteomic, glycome and imaging biomarkers can be used for cancer diagnosis, prognosis and epidemiology. Such biomarkers can be assayed in non-invasively collected biological fluids such as blood, serum, and/or urine. Such biomarkers assayed can include those derived from any type of tissue sample, such as chemically fixed tissue samples. In some examples, tissue samples can include FFPET samples. Biomarkers can be diagnostic (e.g., to identify early stage cancer) and/or prognostic (e.g., to predict how aggressive a cancer will be and/or how a subject will respond to a particular treatment). and/or predict how likely the cancer is to recur).

As used herein, the term "sample" includes a specimen or culture (eg, a microbial culture) that contains or is suspected of containing nucleic acids and/or one or more target nucleic acids. The term "sample" is also meant to include biological, environmental, and chemical samples, as well as any sample for which analysis is desired. A sample can include a specimen of synthetic origin. A sample can contain one or more microorganisms from any source from which one or more microorganisms can be derived. Samples include, but are not limited to, whole blood, skin, serum, plasma, cord blood, chorionic villi, amniotic fluid, cerebrospinal fluid, spinal fluid, lavage fluid (e.g., bronchoalveolar epithelium, stomach, peritoneum, ducts, ears, arthroscopy), tissue samples, chemically fixed samples, FFPET samples, biopsy samples, urine, feces, sputum, saliva, nasal mucus, prostatic fluid, semen, lymphatic fluid, bile, organs, bone marrow, tears, sweat , breast milk, maternal fluids, germ cells, and fetal cells. In some examples, the sample can be fresh or frozen tissue. In some examples, the sample can be a sample that has been treated with a chemical fixative, such as an aldehyde-based fixative. In some examples, the sample can be a formalin-fixed, paraffin-embedded tissue (“FFPET”) sample. In some examples, a sample can be of an in vitro culture established from cells taken from an individual from which nucleic acids can be isolated/purified.

As used herein, the term "target analyte" is intended to mean any analyte to be detected, measured, separated, enriched, isolated, purified, and/or further assayed and analyzed. intended. In some embodiments, the analyte can be any ion, molecule, nucleic acid, biomarker, cell or population of cells, such as, but not limited to, cells of interest, and further assays Its detection, measurement, separation, enrichment and/or use in In some embodiments, the target analyte can be derived from any of the samples described herein.

For the purposes of this disclosure, a given component such as a layer, region, liquid or substrate is herein disposed or formed “on”, “in” or “in” another component. When referred to as a given component, that given component may be directly on another component or may be present on intervening components (e.g., one or more buffer layers, intermediate layers, electrodes or contacts ) may also exist. The terms "disposed to" and "formed on" are used interchangeably to describe how a given component is arranged or arranged with respect to another component. It will be further understood. As such, the terms "disposed on" and "formed on" are not intended to introduce any limitation as to the particular method of material transfer, deposition, or manufacture.

The term "communicate" is used herein to refer to a structural, functional, mechanical, electrical, optical, thermal, or fluid relationship between two or more components or elements, or any of them. used to indicate a combination of Thus, the fact that one component is said to be in communication with a second component implies that additional components can exist between the first and second components, and /or is not intended to exclude the possibility that the first component and the second component can be operably associated or engaged.

As used herein, a "subject" is a mammalian subject (e.g., human, rodent, non-human primate, canine, bovine, ovine, equine, feline, etc.) to be treated and/or Refers to the subject from which the sample is obtained.

"Detecting" a sample within the context of an epitakophoresis device, system or machine can include detecting its location at one, several, or many points throughout the device. Detection can generally be performed by any one or more means that do not interfere with the functioning of the desired device, system or machine and the methods performed using the device, system or machine. In some embodiments, detection includes any means of electrical detection, eg, by detecting conductivity, resistivity, voltage, current, and the like. Furthermore, in some embodiments, detection is electrical detection, thermal detection, optical detection, spectroscopic detection, photochemical detection, biochemical detection, immunochemical detection, and/or chemical detection. any one or more of In some embodiments, one or more target analytes can be detected during ETP-based isolation/purification and optionally collection of said one or more target analytes. Additionally, detection of samples in connection with ETP devices and methods of ETP is disclosed in U.S. Patent Application Publication Nos. 62/585,219 and 62/744,984, and PCT Nos. PCT/EP2018/081049 and PCT/EP2019/077714, the disclosure of which is incorporated herein by reference in its entirety.

In a sample analysis device or system, the term "sample collection volume" refers to the volume of sample intended to be collected, for example by a robotic liquid handler, during or after analysis. In a device for performing epitakophoresis, or a system including such a device, the sample collection volume is the volume intended for collection containing the sample during or after epitakophoresis. In some embodiments, the sample collection volume can be placed in the central well of the devices or systems described herein. In some embodiments, the sample collection volume can be located anywhere that allows collection of the desired sample. In some embodiments, the sample collection volume can be anywhere between the sample loading area and the leading electrolyte electrode/collection reservoir. A sample collection volume can be constituted by any suitable region, vessel, well, or space of a device or system. In some embodiments, the sample collection volume is configured by wells, membranes, compartments, vials, pipettes, and the like. In some embodiments, the sample collection volume can be formed by spaces within or between components of a device or system, such as spaces between two gels or pores in gels.

As used herein, terms such as “ETP device,” “device for performing ETP,” “device for ETP,” are capable of performing ETP or are capable of performing ETP. used interchangeably to refer to a method involving an ETP and/or an apparatus capable of

As used herein, the term "ETP-based isolation/purification" generally refers to devices and methods comprising ETPs, such as devices capable of performing ETPs, such as methods comprising performing ETPs. , ETPs focus one or more target analytes into one or more focusing zones (e.g., one or more ETP bands), thereby extracting one or more target analytes from other materials contained in the initial sample. is isolated/purified. Note that the terms "isolate" and "purify" are used interchangeably. Furthermore, ETP-based isolation/purification generally allows subsequent collection of one or more focused zones (one or more ETP bands) containing the one or more target analytes of interest. The degree of isolation/purification of one or more target analytes provided by one or more ETP-based isolation/purification may be any degree or amount of one or more target analytes from other materials. be able to. In some embodiments, ETP-based isolation/purification of target analytes from a sample is performed, for example, by analytical techniques to determine the composition of an ETP isolation/purification sample containing one or more target analytes. 1% or more, 1% or more, 5% or more, 10% or more, 60% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more of the target analyte when measured; A purity of 40% or greater, 45% or greater, 50% or greater, 55% or greater, 65% or greater, 70% or greater, 85% or greater, 90% or greater, 95% or greater, or 99% or greater can be provided. In some embodiments, the ETP-based isolation/purification of the target analyte from the sample is 1% or less, 1% or more, 5% or more, 10% or more, 15% or more, 20% or more of the target analyte. , 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 85% or more, 90% or more, 95 % or more, or 99% or more recovered from the original sample. In some embodiments, one or more ETP-based isolation/purification can be performed to isolate/purify one or more target analytes, eg, one or more nucleic acids. For example, in some instances, ETP-based isolation/purification is performed on a sample containing one or more target analytes to concentrate the one or more target analytes into one focused zone (ETP band). Focused, one or more target analytes can be substantially separated from other materials contained in the original sample. The sample can be collected after ETP isolation/purification, and the isolated/collected sample can undergo further ETP-based isolation/purification. Optionally, the second ETP-based isolation and purification can be conditioned, in the case of more than one target analyte, to isolate each of the one or more target analytes into separate focusing zones. each of which can optionally be collected separately, thereby separating the target analytes from each other, if desired.

As used herein, the term "mixed sample" generally refers to a sample containing material from more than one source.

As used herein, the term "chemically fixed sample" generally refers to preservation of a sample, such as a tissue sample, through the use of chemical agents well known in the art. For example, a chemically fixed sample can include a sample that has been treated with a cross-linking fixative such as an aldehyde-based fixative. Aldehyde fixatives include those well known in the art, such as formaldehyde fixatives and glutaraldehyde fixatives. In some examples, chemically fixed samples can include FFPET samples.

As used herein, the term "FFPET sample solution" generally refers to, for example, a device for performing ETP to isolate/purify one or more nucleic acids contained in an FFPET sample. refers to a FFPET sample in solution form, such as a solution containing a dissolved FFPET sample, in which

As used herein, the terms "sample pretreatment,""pretreatedsample," etc. generally refer to any procedure performed on a sample prior to loading the sample into the ETP device. Point. For example, sample pretreatment can include preparing an FFPET sample solution from an FFPET sample, such as by dissolving FFPET curls under conditions suitable for FFPET curl dissolution. Such methods are known in the art and include, for example, the lysis protocol used as part of the KAPA Express Extract method. For example, a lysis buffer may be added to the FFPET curl, followed by heating the solution until the desired solubility is achieved.
Method and apparatus

As noted above, current approaches to extracting, isolating, and/or purifying nucleic acids from stored specimens, such as chemically fixed samples, such as FFPET samples, have many drawbacks. General procedures for isolating nucleic acids from FFPET may include; removal of paraffin (deparaffinization), lysis of stored samples (protease digestion), reversal of crosslinks acquired during the fixation process. , and purification of nucleic acids. These protocols are typically complex and labor intensive and result in small amounts of low quality nucleic acids. For example, nucleic acids obtained from FFPET samples using current approaches are often of poor quality and low quantity. For example, RNA isolated from FFPET samples is often moderately to highly degraded and fragmented, a significant drawback to its use in diagnostic and/or therapeutic applications. Furthermore, conventional techniques for extraction, isolation and/or purification of nucleic acids from samples that have been subjected to chemical fixation, including the use of aldehyde-based fixatives, often involve the use of organic solvents such as xylene. Including the use of harsh chemicals. Conventional techniques also typically use column and/or bead-based processes that are costly, labor intensive, and poorly suited to automation. To solve such problems, the present disclosure generally describes an apparatus and method for analysis of a sample containing one or more nucleic acids, e.g. performing epitachophoresis to isolate and/or purify the nucleic acid, wherein the isolated and/or purified nucleic acid is optionally subjected to further downstream assays such as in vitro diagnostic (“IVD”) assays. can be served to

Furthermore, although the devices and methods of the present invention allow extraction of total genomic and/or total nucleic acid content from samples and/or biological samples, such extraction may be performed using conventional capillary or microfluidic techniques. Difficulties may arise when using ITP-based devices and methods, particularly ITP-based capillary or microfluidic devices and methods. Furthermore, the highly efficient extraction of target nucleic acids obtained by use of the devices and methods described herein can be achieved in downstream in vitro diagnostic (IVD) assays where the amount of target nucleic acids, e.g., DNA and/or RNA This downstream IVD method directly correlates with the sensitivity that can be measured. Sometimes spin columns or magnetic glass particles that bind nucleic acids on their surface can be conventionally used to perform nucleic acid extraction. Compared to these conventional techniques, the devices and methods described herein can provide any one or more of the following advantages: Higher extraction yield (potentially less loss); simpler instrument setup compared to the larger footprint of benchtop instruments; potentially faster performance compared to other instruments applied to similar applications sample turnaround and high parallelizability; easy integration with other microfluidic-based systems for downstream processing of extracted nucleic acids. In some embodiments, a nucleic acid obtained by ETP-based isolation/purification of a sample, eg, a FFPET sample, can include the total nucleic acid content of the sample, eg, both DNA and RNA from the sample. In some examples, methods involving ETP-based isolation/purification may involve simultaneous collection of nucleic acids, including DNA and RNA, wherein the collected nucleic acids are subjected to further downstream assays, such as any known in the art. can be subjected to a method of separating DNA and RNA for the separation of DNA and RNA by means of

Further, the present disclosure generally relates to methods of isolating and/or purifying one or more nucleic acids from a sample, such as a chemically fixed sample, the method comprising: a. providing a device for performing epitakophoresis (ETP); b. providing a sample comprising said one or more nucleic acids; c. performing one or more epitachophoresis runs by using the device to perform ETP to focus the one or more nucleic acids into one or more focusing zones, e.g., as one or more ETP bands; and d. collecting said one or more nucleic acids by collecting said one or more focused zones containing said one or more nucleic acids; thereby obtaining one or more isolated and/or purified nucleic acids and optionally said sample comprises a FFPET sample solution comprising said nucleic acid.

In some embodiments, a method of isolating and/or purifying one or more nucleic acids from a sample, eg, a chemically fixed sample, comprises a. providing a device for performing epitakophoresis (ETP); b. providing an FFPET sample containing nucleic acids; c. preparing an FFPET sample solution from said FFPET sample; d. performing one or more epitachophoresis runs by performing ETP using the device to focus the one or more nucleic acids into one or more focusing zones, e.g., as one or more ETP bands; and e. collecting said one or more nucleic acids by collecting said one or more focused zones containing said one or more nucleic acids; thereby producing one or more isolated and/or purified nucleic acids; including obtaining In some examples, nucleic acids can include DNA and/or RNA.

In some examples, samples for ETP-based sample analysis can include any type of formaldehyde-crosslinked biological sample. In some embodiments the sample may comprise a tissue sample, optionally the tissue sample comprises animal tissue. In some embodiments, the sample may comprise a paraffin-embedded sample. For example, the sample can be formalin-fixed, paraffin-embedded tissue (FFPET). In some embodiments, a sample is obtained from an animal (e.g., human) and may then be stored in a formaldehyde-containing solution to stabilize the sample prior to analysis, thereby removing nucleic acids in the sample. and/or crosslink proteins.

In some embodiments, the method for ETP-based isolation and/or purification of one or more nucleic acids can be automated, eg, by using an automated ETP system. For example, US Pat. Nos. 62/585,219 and 62/744,984, and PCT Nos. PCT/EP2018/081049 and PCT/EP2019/077714, the disclosures of which are incorporated herein by reference in their entirety. incorporated).

In some embodiments, ETP devices for use with the methods described herein are described in US Pat. No. 62/585,219 and US Pat. /EP2018/081049 and PCT/EP2019/077714, the disclosures of which are incorporated herein by reference in their entirety.

In some embodiments, the method may comprise ETP-based isolation and/or purification of one or more nucleic acids from one or more FFPET samples, wherein the nucleic acids comprise DNA and/or RNA. In some examples, the method may result in isolating and/or purifying both DNA and RNA at one or more ETP bands and/or focal zones during a single ETP run. In some examples, after ETP-based isolation and/or purification, RNA and DNA can be separated from each other and subjected to one or more IVD assays, sequencing, and/or gene expression analysis. It can be subjected to further downstream assays such as profiling. In some embodiments, the FFPET sample can include FFPET curls, which are then treated to produce FFPET sample solutions that can optionally be loaded into the ETP device.

In some embodiments, devices and/or methods for epitacophoresis can allow for the focusing and collection of target analytes for any desired amount of time that allows for the desired focusing and collection. . In some embodiments, the method can include performing ETP for 120 minutes or more, 120 minutes or less, 100 minutes or less, 80 minutes or less, 60 minutes or less, 50 minutes or less, or 40 minutes or less.

In some embodiments, nucleic acids obtained by ETP-based isolation/purification of nucleic acids from FFPET samples are isolated using conventional techniques, such as those described above, e.g., bead-based and/or column-based methods. It may be of higher yield and/or higher quality compared to nucleic acids obtained from FFPET samples. In some embodiments, nucleic acids obtained by ETP-based isolation/purification of nucleic acids from one or more FFPET samples are measured by qPCR-based analysis (e.g., quality control (qc) qPCR, etc.). Q-scores obtained from qPCR-based analysis) can be of equal or higher quality. Note that Q-scores range from 0 (poor quality) to 1 (high quality), with higher quality samples generally preferred for downstream IVD applications such as sequencing-based applications. In some embodiments, a 1.25-fold increase using methods comprising ETP-based isolation/purification of nucleic acids from one or more FFPET samples compared to bead-based and/or column-based methods 1.5 times or more, 1.75 times or more, 2.0 times or more, 2.25 times or more, 2.5 times or more, 2.75 times or more, 3 times or more, 4 times or more, 5 times or more, 10-fold or more, 100-fold or more, or 1000-fold or more nucleic acids can be obtained. In some embodiments, the amount of isolated and/or purified nucleic acid obtained from the methods described herein can be any amount, depending on the sample used. can be at least partially present for In some examples, the amount of isolated and/or purified nucleic acid can range anywhere from sub-nanograms to macrograms or more.

In some embodiments, the electric field strength can be from about 10 V to about 10 kV at powers ranging from about 1 mW to about 100 W within a convenient time frame for moving charged particles in the present methods and apparatus. In some embodiments, the maximum power applied for the fastest analysis may depend on the electrical resistivity of the sample and electrolyte solution, as well as the cooling capabilities of the materials that may be used in constructing the devices described herein.

In some embodiments, the ETP-based isolation and/or purification of one or more nucleic acids from one or more chemically fixed samples is 1% of the one or more nucleic acids contained in the original sample. 1% or more, 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, It can result in isolation and collection of 60% or more, 65% or more, 70% or more, 85% or more, 90% or more, 95% or more, or 99% or more. In some embodiments, the method provides an isolated/purified one or more nucleic acids, e.g., as measured by an analytical technique for determining the composition of an ETP isolated/purified sample comprising one or more nucleic acids. 1% or less, 1% or more, 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% of the above nucleic acids Purity of 55% or more, 60% or more, 65% or more, 70% or more, 85% or more, 90% or more, 95% or more, or 99% or more can be provided. In some embodiments, one or more buffer concentrations, e.g., LE and/or TE buffer concentrations, percentage of gel contained in the ETP device, and/or stop time for ETP-based isolation and collection runs are , may be modified and/or optimized to facilitate separation of the one or more nucleic acids from other materials contained in the sample.

In some embodiments, one or more nucleic acids isolated/purified by ETP-based isolation/purification can be of any desired size. In some embodiments, the nucleic acid is 5 nt or less, 10 nt or less, 20 nt or less, 30 nt or less, 50 nt or less, 100 nt or less, 1000 nt or less, 10,000 nt or less, 100,000 nt or less, 1,000,000 nt or less, or 1 ,000,000 nt or larger. In some embodiments, the method can further comprise detecting the one or more nucleic acids during and/or after the ETP-based isolation and/or purification, e.g., detecting Some examples include optical detection, wherein the optical detection includes detection of intercalating dyes and/or optical labels bound to and/or associated with the one or more nucleic acids. In some embodiments, the detection can include electrical detection, such as voltage monitoring. In some embodiments, the detection includes monitoring movement of a dye, such as brilliant blue, and adjusting any one or more ETP parameters, such as starting or stopping sample collection, based on the movement of the dye. and

In some embodiments, the method of ETP-based isolation/purification of one or more nucleic acids, optionally from one or more FFPET samples, is such that the samples are automatically loaded into the device and/or the It can be an automated method in which one or more nucleic acids are automatically collected from the device. In some embodiments, the isolated and/or purified one or more nucleic acids are subjected to one or more further ETP runs to further isolate and/or purify the one or more nucleic acids. can be

In some embodiments, the method may further comprise the use of an ETP top marker, such as those described in US Patent Application No. 62/847,699, which is hereby incorporated by reference in its entirety. In some embodiments, isolated and/or purified nucleic acids can further undergo CCancer personalized profiling by deep sequencing (CAPP-Seq). In some embodiments, isolated and/or purified nucleic acids can be assayed for one or more biomarkers. In some embodiments, the isolated and/or purified nucleic acid can be further evaluated in one or more assays to identify both CNVs and infectious agents. In some embodiments, isolated and/or purified nucleic acids, quantitative and qualitative analysis of nucleic acids such as DNA strand integrity, mutation frequencies, microsatellite aberrations, and gene methylation. can be further evaluated by one or more methods of detecting specific tumor-specific changes. In some embodiments, the isolated and/or purified nucleic acids are further evaluated by one or more methods, e.g., to detect diagnostic, prognostic, and monitoring markers in samples from cancer patients. can be In some embodiments, the method can be further combined with CNV detection to provide a method to aid in clinical diagnosis, treatment, outcome prediction and progression monitoring in patients with or suspected of having malignancies. In some embodiments, isolated and/or purified nucleic acids are used to detect genetic features in a sample, including copy number variations (CNVs), insertions, deletions, translocations, polymorphisms and mutations. can be further provided for a method for In some embodiments, the concentration of any one or more of the isolated and/or purified one or more nucleic acids can be determined. In some embodiments, concentration can be determined by molecular barcoding. In some embodiments, the isolated and/or purified nucleic acids can be further analyzed for tumor-derived SNVs. In some embodiments, nucleic acids can include nucleic acids derived from one or more cancerous cells.

Furthermore, the present disclosure generally provides a method of identifying a tumor-derived SNV, comprising (a) a sample from a subject having or suspected of having cancer (optionally, the sample is an FFPET sample); (e.g., an FFPET sample solution); and (b) performing ETP-based isolation and/or purification to isolate and/or purify the target nucleic acid to provide an isolated and/or purified sample. (c) performing a sequencing reaction on the isolated and/or purified sample to generate sequencing information; and (d) the sequencing information from step (c). applying an algorithm to the sequencing information to form a list of candidate tumor alleles based on the candidate tumor alleles containing non-dominant bases that are not germline SNPs; ) identifying tumor-derived SNVs based on the list of candidate tumor alleles. In some embodiments, candidate tumor alleles can include genomic regions that contain candidate SNVs.

Still further, the present disclosure is generally directed to methods of identifying viral-derived nucleic acids, comprising: (a) a sample, e.g., a chemically fixed sample, from a subject suspected of having a viral infection or having been exposed to a virus; (b) performing an ETP-based isolation and/or purification to isolate and/or purify the target nucleic acid to obtain an isolated and/or purified sample; (c) performing a sequencing reaction on the isolated and/or purified sample to generate sequencing information; and (d) based on the sequencing information, the subject is infected with one or more viruses. and determining whether the

Furthermore, in further exemplary embodiments, the device for sample analysis described herein comprises Dimensions can be included to accommodate more than one sample volume. In some embodiments, the concentration of one or more isolated and/or purified nucleic acids can be measured. In some embodiments, the sample volume of the sample loaded into the ETP device for ETP-based isolation/purification is 0.25 mL or less, 0.25 mL or more, 0.5 mL or more, 0.75 mL or more, 1 0 mL or greater, 2.5 mL or greater, 5.0 mL or greater, 7.5 mL or greater, 10.0 mL or greater, 12.5 mL or greater, or 15.0 mL or greater.

In further exemplary embodiments, the device can be used to concentrate target analytes, for example, from about 2-fold to about 1000-fold or more. In some embodiments, the target analyte may comprise one or more nucleic acids. In further embodiments, the target analytes can include small inorganic and organic ions, peptides, proteins, polysaccharides, DNA, or microorganisms such as bacteria and/or viruses.

In some embodiments, the nucleic acids collected by ETP-based isolation/purification can be used for one or more downstream in vitro diagnostic applications. Furthermore, in some embodiments, the ETP device for sample analysis is, for example, a capillary analyzer, a chromatograph, a PCR device, an enzymatic reactor, and/or other devices to perform further sample analysis. It can be connected online to any other device that can be used, for example devices related to IVD applications. In some embodiments, ETP devices can be used in workflows involving the preparation of nucleic acid sequencing libraries. Further, in some embodiments, the ETP device is used with a liquid handling robot that can optionally be used to perform downstream analysis of samples that may have been focused and/or collected from the device. can be done.

In some embodiments, the method may comprise ETP-based isolation and purification of one or more nucleic acids, including DNA and/or RNA, wherein the nucleic acids are isolated from one or more biological samples. / refined. Such samples include, but are not limited to, fresh samples or cell/tissue aspirates, frozen sections, needle biopsies, cell cultures, fixed tissue samples, cell buttons, tissue microarrays, and the like. In some embodiments, the sample comprises a fixed paraffin-embedded tissue (eg, FFPET) sample. Histological samples are typically fixed with aldehyde fixatives such as formalin (formaldehyde) and glutaraldehyde, although the methods described herein use other fixing techniques such as alcohol immersion. It can also be performed with fixed tissue. In some examples, samples may include biopsies and fine needle aspirates and archival samples (eg, tissue microarrays), and the like. In some examples, samples can include, but are not limited to, FFPET samples from human tissue, laboratory animal tissue, companion animal tissue, or livestock animal tissue. Thus, for example, samples include, but are not limited to, samples from healthy humans (e.g., healthy human tissue samples), samples from diseased subjects and/or diseased tissues, used in diagnostic and/or prognostic assays. Examples include tissue samples from humans, including samples such as those obtained from human tissue. Suitable samples also include samples from non-human animals. For example, non-human primates such as chimpanzees, gorillas, orangutans, gibbons, monkeys, macaques, baboons, mangabeys, colobus, langurs, marmosets, lemurs, mice, rats, rabbits, guinea pigs, hamsters, cats, ferrets, fish, cows, FFPET samples from pigs, sheep, goats, horses, donkeys, chickens, geese, ducks, turkeys, amphibians, or reptiles can be used in the methods described herein.

Further, in some embodiments, FFPET samples of any age can be used in the methods described herein, including fresh, less than 1 week, less than 2 weeks, less than 1 month, 2 months less than 3 months, less than 6 months, less than 9 months, less than 1 year, at least 1 year, at least 2 years, at least 3 years, at least 4 years, at least 5 years, at least 6 years, at least 7 years, Including, but not limited to, FFPET samples that are at least 8 years old, at least 9 years old, at least 10 years old, at least 15 years old, at least 20 years old, or older.

In some embodiments, a fixed embedded tissue sample (eg, an FFPET sample) comprises regions of diseased tissue, such as tumors or other cancerous tissue. Such FFPET samples find utility in the methods described herein, but do not contain regions of diseased tissue, e.g., FFPET samples from normal, untreated, placebo-treated, or healthy tissue. can also be used in the methods described herein. In some embodiments of the methods described herein, a desired diseased area or tissue, or a region containing a particular region, feature or structure within a particular tissue, is the percentage of nucleic acid obtained from the desired region is identified in the FFPET sample, or one or more sections thereof, prior to isolation of nucleic acids as described herein, to increase . Such regions or regions can be identified using any method known to those of skill in the art, including, but not limited to, visual identification, staining such as hematoxylin and eosin staining, immunohistochemical labeling, and the like. can. In any event, in some embodiments, a desired amount of tissue sample or section thereof is used to obtain starting material for ETP-based isolation/purification of nucleic acid samples using the methods described herein. Regions can be dissected by either macrodissection or microdissection.

In certain exemplary but non-limiting embodiments, the sample comprises a diseased area or tissue containing cancer-derived cells. In some embodiments, the cancer is acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), adrenocortical carcinoma, AIDS-related cancers (e.g., Kaposi's sarcoma, lymphoma), anal cancer, appendiceal cancer, Astrocytoma, atypical teratoid/rhabdomyosarcomatous tumor, cholangiocarcinoma, extrahepatic carcinoma, bladder cancer, bone cancer (e.g., Ewing's sarcoma, osteosarcoma, malignant fibrous histiocytoma), brainstem glia tumor, brain tumor (e.g., astrocytoma, brain and spinal cord tumor, brain stem glioma, central nervous system atypical teratoid/rhabdomyosarcomatous tumor, central nervous system embryonic tumor, central nervous system germ cell tumor , craniopharyngioma, ependymoma, breast cancer, bronchial tumor, Burkitt's lymphoma, carcinoid tumor (e.g. childhood, gastrointestinal), cardiac tumor, cervical cancer, chordoma, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), chronic myeloproliferative disease, colon cancer, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, ductal carcinoma, e.g. (biliary, extrahepatic), ductal carcinoma in situ (DCIS), embryonic tumor, endometrial cancer, ependymoma, esophageal cancer, sensory neuroblastoma, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic cholangiocarcinoma, eye cancer (e.g., intraocular melanoma, retinoblastoma), Fibrous histiocytoma of bone, malignant, osteosarcoma, gallbladder cancer, gastric (stomach) cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors (GIST), germ cell tumors (e.g. ovarian cancer) , testicular cancer, extracranial cancer, extragonadal cancer, central nervous system), gestational chorioblastic tumor, brain stem cancer, hairy cell leukemia, head and neck cancer, heart cancer, hepatocellular (liver) cancer, histiocytosis, Langerhans Cellular carcinoma, Hodgkin's lymphoma, hypopharyngeal carcinoma, intraocular melanoma, islet cell tumor, pancreatic neuroendocrine tumor, Kaposi's sarcoma, renal cancer (e.g. renal cell, Wilms tumor, and other renal tumors), Langerhans cell histiocytosis , laryngeal cancer, leukemia, acute lymphoblastic (ALL), acute myelogenous (AML), chronic lymphocytic (CLL), chronic myelogenous (CML), hair cell, lip and oral cancer, liver cancer (primary ), lobular carcinoma in situ (LCIS), lung cancer (e.g., childhood, non-small cell, small cell), lymphoma (e.g., AIDS-related), Burkitt (e.g., non-Hodgkin's lymphoma), cutaneous T cells (e.g., mycosis fungoides, Sézary syndrome), Hodgkin, non-Hodgkin, primary central nervous system (CNS)), macroglobulinemia, Waldenstrom, male breast cancer, malignant fibrous histiocytoma and osteosarcoma of bone, melanoma (e.g. childhood, intraocular (eye)), Markle cell carcinoma, mesothelioma, metastatic squamous neck cancer, midline tract carcinoma, oral cancer, multiple endocrine Neoplastic syndrome, multiple myeloma/plasma cell tumor, mycosis fungoides, myelodysplastic syndrome, myeloid leukemia, chronic (CML), multiple myeloma, nasal and sinus cancer, nasopharyngeal carcinoma, neuroblast cancer, oral cavity cancer, lip and oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, pancreatic neuroendocrine tumor (pancreatic islet cell tumor), papilloma, paraganglioma, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer , pharyngeal carcinoma, pheochromocytoma, pituitary tumor, plasma cell tumor, pleuropulmonary blastoma, primary central nervous system (CNS) lymphoma, prostate cancer, rectal cancer, renal cell (kidney) carcinoma, renal pelvis and ureter, migration sex cell carcinoma, rhabdomyosarcoma, salivary gland carcinoma, sarcoma (e.g., Ewing, Kaposi, osteosarcoma, rhabdomyosarcoma, soft tissue, uterine), Sézary syndrome, skin cancer (e.g., melanoma, Merkle cell carcinoma, basal cell carcinoma, non-melanoma), small bowel cancer, squamous cell carcinoma, cervical squamous cell carcinoma of unknown primary, stomach (gastric) cancer, testicular cancer, throat cancer, thymoma and thymic carcinoma, thyroid cancer , trophoblastic tumor, ureteral and renal pelvis cancer, urethral cancer, uterine cancer, endometrial cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom's macroglobulinemia, Wilm's tumor, etc. Including cancer.

The apparatus and methods illustratively disclosed herein may be practiced in the absence of any element specifically disclosed herein and/or any element specifically disclosed herein. can be properly implemented.

Example 1: Device for epitachophoresis

Devices for epitakophoresis commonly use concentric or polygonal disk architectures, eg, as shown in FIGS. 1-4. Glass or ceramic are used in the manufacture of the system (ie concentric or polygonal disk material) because these materials provide improved heat transfer properties which are beneficial during device operation. For example, flat channels in an epitakophoresis device have better heat transfer capabilities compared to narrow channels, so overheating (or boiling) of the focused material is generally prevented. Current/voltage programming is also suitable for adjusting the Joule heating of the device. Plastic materials are also used in device manufacturing. Generally, the device is manufactured with dimensions to accommodate a desired sample volume, eg, a milliliter-scale sample volume, eg, up to 15 mL.

Referring to FIGS. 1-3, two concentric discs are separated by a spacer, thereby forming a flat channel for epitakophoretic sample processing. Current is applied through a plurality of high voltage connections (HV connections) and the central ground connection of the system (see, eg, FIGS. 1 and 3). In some examples, the sample is injected into the device through an opening in the device, such as the top or side (see, eg, FIG. 3). Application of electricity focuses the target analytes of the sample as concentric rings moving to the center of the disc (discussed further below), which are then collected through a syringe at the bottom of the device (e.g., FIG. 3 (see ). As shown in FIGS. 2A (top view) and 2B, an example device configuration includes an outer circular electrode (1), a terminating electrolyte (2), and a leading electrolyte (3). Generally, the diameter of the outer circular electrode (1) is about 10-200 mm and the diameter of the preceding electrolyte ranges in thickness (height) from about 10 μm to about 20 mm. The leading electrolyte may be stabilized by gels, viscous additives, or may be hydrodynamically separated from the terminating electrolyte by, for example, a membrane. A gel or hydrodynamic separation prevents mixing of the leading and terminating electrolytes during operation of the device. Also, in some devices, mixing is prevented by using a very thin (<100 μm) layer of electrolyte, as described further below in Example 2.

Referring to Figures 2A-2B, in the center of the precursor electrolyte is an electrode reservoir (4) with an electrode (5). The assembly of electrodes (1,5) and electrolytes (2,3) is placed on a flat electrically insulating support (8). The electrolyte reservoir (4) is used to remove the concentrated sample solution after the separation process, eg pipetting the sample out of the reservoir.

In an alternative configuration (see Figure 4) the center electrode (5) is moved to a preceding electrolyte reservoir (10) connected to the concentrator by a tube (9). The tube (9) is directly or closed at one end by a semi-permeable membrane (not shown). This arrangement facilitates collection by stopping the migration of large molecules according to the properties of the membrane used. This arrangement simplifies sample collection and provides a means of connecting the concentrator on-line to other devices such as capillary analyzers, chromatography, PCR devices, enzymatic reactors, and the like. A tube (9) can also be used to provide a countercurrent flow of the preceding electrolyte in a configuration that does not contain a gel containing the preceding electrolyte.

Generally, gels for prior electrolyte stabilization are formed by any non-charged material such as agarose, polyacrylamide, pullulan, and the like. In some devices the top surface is left open, or in some devices the top surface is closed, depending on the nature of the separation to be performed. When closed, the material used to cover the device is preferably a thermally conductive insulating material to prevent evaporation during operation of the epitakophoresis device.

Generally, the ring (circular) electrode is preferentially a gold or platinum plated stainless steel ring to allow for maximum chemical resistance and field uniformity. Alternatively, stainless steel and graphite electrodes may be used in some devices, especially disposable devices. Also, the ring (circular) electrodes can be replaced by other parts that serve a similar function, such as an array of wire electrodes. Furthermore, a two-dimensional array of regularly spaced electrodes may additionally or alternatively be used in an epitakophoresis device. An array of regularly spaced electrodes in a circular orientation can also be used in an epitakophoresis device. Furthermore, other electrode configurations can be used to provide different electric field shapes based on the desired sample separation (eg, to direct the focus zone). Such a configuration is described as a polygonal arrangement of electrodes. When divided into electrically isolated segments, a switched electric field is produced for the time-dependent shape of the driving electric field. Such an arrangement facilitates sample collection in some devices.

Example 2: Operation of the epitakophoresis device

Epitachophoresis devices, such as those of the designs shown in FIGS. 1-4, have two electrolyte reservoir arrangements, a leading electrolyte followed by a sample mixed with a terminating electrolyte, as shown in FIG. or a sample mixed with a leading electrolyte followed by a terminating electrolyte, or operated with either a three electrolyte reservoir arrangement. In such a configuration the sample can be mixed with any conductive solution. Alternatively, the terminating electrolyte zone can be eliminated if the sample contains appropriate terminating ions. Referring to FIGS. 2A-2B, when the termination electrolyte reservoir (2) is filled with a mixture of sample and appropriate termination electrolyte and the power supply (6) is turned on, the ions migrate towards the central electrode (5). First, a zone is formed at the boundary between the leading electrolyte and the terminating electrolyte (7). The concentration of the sample zone during migration is determined according to the general principle of isotonicity [Foret, F. , Krivankova, L.; , Bocek, P.; , Capillary Zone Electrophoresis. Electrophoresis Library, (Editor Radola, BJ) VCH, Verlagsgessellschaft, Weinheim, 1993. ]. As a result, low-concentration sample ions are concentrated and high-concentration sample ions are diluted. When the sample zone enters the electrolyte reservoir (4) the separation process is stopped and the focused material is collected in the center of the device. In practice, the final concentration of the migration zone has a concentration comparable to that of the leading ions. Typically, any concentration factor from 2 to 1000 or more is achieved using epitakophoresis.

In a three-electrolyte reservoir arrangement, the sample is applied between the leading and terminating electrolytes (see, for example, FIG. 5), and such an arrangement provides slightly faster sample concentration compared to the two-electrolyte reservoir arrangement. and cause separation.

To avoid mixing, the leading and trailing electrolytes are placed in a neutral (uncharged) viscous medium, e.g., an agarose gel (e.g., optionally contained within a gel or hydrodynamically separated from the terminating electrolyte). 2A-2B, see FIG. 3) representing the preceding electrolyte.

All common electrolytes known to those skilled in the art for use in isotachophoresis can be used in accordance with the present invention, provided that the leading ions have an effective electrophoretic mobility higher than that of the sample ions of interest. epitachophoresis device. The opposite is true for selected terminal ions.

The device is operated in either positive mode (separation/concentration of cationic species) or negative mode (separation/concentration of anionic species). The most common precursor electrolytes for anion separation using epitacophoresis are, for example, chloride, sulfate, or formate buffered to the desired pH with a suitable base, such as histidine, TRIS, creatinine, etc. include. Concentrations of precursor electrolytes for epitacophoresis for anion separation range from 5 mM to 1 M for the major ion. In that case, terminal ions often include MES, MOPS, HEPES, TAPS, acetate, glutamate and other anions of weak acids and low mobility anions. Terminal electrolyte concentrations for positive mode epitachophoresis range from 5 mM to 10 M for terminal ions.

For cation separation, common leading ions for epitacophoresis include, for example, potassium, ammonium or sodium, with acetate or formate being the most common buffering counterions. The reactive hydroxonium ion migration boundary then functions as a universal terminating electrolyte formed by any weak acid.

In both positive and negative modes, increasing the concentration of precursor ions results in a proportional increase in sample zone at the expense of an increase in current (power) for a given applied voltage. Typical concentrations range from 10-100 mM; however, higher concentrations are possible.

Furthermore, if zone electrophoretic separation alone is sufficient, the device can be operated with only one background electrolyte.

Current and/or voltage programming are suitable for adjusting the movement speed of the sample. Note that in this concentric arrangement the cross-sectional area changes during movement and the speed of zone movement is not constant over time. This arrangement therefore does not strictly follow the principle of isotachophoresis, in which the zones move at a constant speed. Depending on the operating mode of the power supply (6), three basic cases can be distinguished:1. galvanostatic separation;2. constant voltage separation;3. Constant current isolation.

The variables in the equations described below are: d = distance traveled (d<0; r>); E = electric field strength; H = electrolyte (gel) height; I = current; J = current density; κ=electrolyte conductivity; r=radius; S=cross-sectional area; u=electrophoretic mobility; v=velocity;

In a common mode of operation using constant current supplied by a high voltage power supply (HVPS), the moving region accelerates as it approaches the center due to the increase in current density. For constant current separation, using a device containing a circular structure, such as one or more circular electrodes, the relative velocity at distance d is the epitachonic velocity at v at distance d from starting radius r as It depends only on the mobility (conductivity) of the preceding electrolyte, as demonstrated by the derivation of the foresis boundary velocity.

General formula:

Epitachophoresis boundary velocity v at distance d of radius r from the start:

See FIG. 6B for a plot of distance traveled (d) versus relative velocity at distance d at constant current.

For separation at constant voltage, using a device with a circular structure, for example a device with one or more circular electrodes, the relative velocity at distance d is given by: It depends on the mobility (conductivity) of both LE and TE, as demonstrated by the derivation of the tachophoresis boundary velocity:

General formula:

Boundary velocity calculation:

See FIG. 6C for a plot of distance traveled (d) versus relative velocity at distance d at constant voltage.

For constant power separation, using a device with a circular structure, for example a device with one or more circular electrodes, the relative velocity at distance d is given by: It depends on the mobility (conductivity) of both LE and TE, as demonstrated by the derivation of the tachophoresis boundary velocity:

General formula:

Boundary velocity calculation:

κ is a small number, so:

See FIG. 6D for a plot of distance traveled (d) versus relative velocity at distance d at constant power.

Example 3: Epitachophoresis using an exemplary device

As shown in FIG. 7, an epitakophoretic separation was performed by focusing the sulfanilic acid dye (SPADNS) into concentric rings using an epitakophoretic apparatus. Epitachophoresis was performed in the epitakophoresis apparatus by applying a constant power of 1 W.

Referring to FIG. 7, the SPADNS was focused into concentric ring-like focusing zones that can be seen as red zones in FIG. The top half of the red circle indicated that the zone height was approximately 5 mm. As the epitakophoresis zone moves from the edge of the device towards the center, eventually the SPADNS focusing zone enters the center of the device and is collected in the center of the device, thereby providing the desired epitakophoresis using epitakophoresis. Sample focusing and recovery was demonstrated.

Example 4: Epitachophoresis using an exemplary device

Epitacophoresis was performed to focus sulfanilic acid (SPADNS) using an epitakophoresis apparatus (Fig. 8A). The device of Figure 8A had a circular configuration and a circular gold electrode with a diameter of 10.2 cm. HCl-Histidine (pH 6.25) was used as the pre-electrolyte and contained in 10 mL of 0.3% agarose gel with a diameter of 5.8 cm. 15 mL of MES Tris (pH 8.00) was used as trailing electrolyte. The syringe reservoir of the device contained the pre-electrolyte His (pH 6.25). 300 μl of SPADNS at a concentration of 0.137 mM was prepared in trailing electrolyte and loaded into the device. A constant power of 1 W was used to perform epitachophoresis.

Referring to FIG. 8B, the SPADNS was focused into concentric ring-like focusing zones that can be seen as red zones in FIG. 8B. As the epitakophoresis zone moves from the edge of the device towards the center, eventually the SPADNS focusing zone enters the center of the device and is collected in the center of the device, thereby providing the desired epitakophoresis using epitakophoresis. Sample focusing and recovery was demonstrated.

Furthermore, epitakophoresis was performed using the epitakophoresis apparatus of FIG. 8A to focus 30 nt oligomers (ROX-oligos). The device of Figure 8A had a circular configuration and a circular gold electrode with a diameter of 10.2 cm. 10 mM HCl-histidine (pH 6.25) was used when the pre-electrolyte was contained in 10 mL of 0.3% agarose gel with a diameter of 5.8 cm. 15 mL of 10 mM MES Tris (pH 8.00) was used as trailing electrolyte. The device syringe reservoir contained the pre-electrolyte His (pH 6.25) at a concentration of 100 mM. 75 μl of ROX-oligos at a concentration of 100 μm were prepared in trailing electrolyte and loaded into the device. A constant power of 1 W was used to perform epitachophoresis.

Referring to FIG. 8C, the ROX oligos were focused into concentric focusing zones that can be seen as blue zones in FIG. 8C. As the epitakophoresis zone moved from the edge of the device towards the center, eventually the ROX-oligo focused zone entered the center of the device and was collected in the center of the device, thereby using epitakophoresis. Focusing and recovery of the desired sample was demonstrated.

Example 5: Epitachophoresis using an exemplary device

Epitacophoresis was performed using an epitakophoresis device (FIGS. 9A-9B) to focus the sulfanilic acid dye (SPADNS), which was then collected from the device (FIGS. 9C-9D). The device of Figures 9A-9B had a circular construction and a circular stainless steel wire electrode with a diameter of 11.0 cm. Referring to FIG. 9B, the numbers in the schematic represent dimensions in millimeters. 20 mM HCl-histidine (pH 6.20) was used as the pre-electrolyte. 5 mL of 10 mM MES Tris (pH 8.00) was used as trailing electrolyte with a 0.3% agarose gel in LE, the gel having a diameter of 8.9 cm (Fig. 9C) and formed prior to introduction of TE. or 15 mL of 10 mM MES Tris (pH 8.00) was used as trailing electrolyte contained in a 0.3% gel with a diameter of 5.8 cm (Fig. 9D) and formed prior to the introduction of TE. bottom. The electrode reservoir of the device contained the pre-electrolyte His (pH 6.25) at a concentration of 100 mM.

Referring to FIG. 9C, 150 μl of SPADNS at a concentration of 0.137 mM was prepared in 15 mL of trailing electrolyte and loaded into the device. A constant power of 2 W was used to perform epitachophoresis. The SPADNS was focused into concentric ring-like focusing zones that can be seen as red zones in FIG. 9C. As the epitakophoresis zone moves from the edge of the device towards the center, eventually the SPADNS focusing zone enters the center of the device and is collected in the center of the device, thereby providing the desired epitakophoresis using epitakophoresis. Sample focusing and recovery was demonstrated. The recovered SPADNS had a 40-fold increase in absorbance compared to the absorbance of the initial 15 mL SPADNS-containing sample.

Referring to FIG. 9D, 150 μl of SPADNS at a concentration of 0.137 mM was prepared in 15 mL of trailing electrolyte and loaded into the device. A constant power of 2 W was used to perform epitachophoresis. The SPADNS was focused into concentric ring-like focusing zones that can be seen as red zones in FIG. 9D. As the epitakophoresis zone moves from the edge of the device towards the center, eventually the SPADNS focusing zone enters the center of the device and is collected in the center of the device, thereby providing the desired epitakophoresis using epitakophoresis. Sample focusing and recovery was demonstrated. The recovered SPADNS had a 40-fold increase in absorbance compared to the absorbance of the initial 15 mL SPADNS-containing sample.

Using the epitakophoresis device of Figures 9A-B, epitakophoresis was performed to focus SPADNS from saline in the gel-free device. 20 mM HCl-histidine (pH 6.20) was used as the pre-electrolyte. 13 mL of 10 mM MES Tris (pH 8.00) was used as trailing electrolyte, which was further mixed with 3 mL of 0.9% NaCl. The electrode reservoir of the device contained the pre-electrolyte HCl histidine (pH 6.25) at a concentration of 100 mM.

Referring to FIG. 10, 150 μl of SPADNS at a concentration of 0.137 mM was prepared in 13 mL of trailing electrolyte mixed with 3 mL of 0.9% NaCl and loaded into the device. A constant power of 2 W was used to perform epitachophoresis. The SPADNS was focused into concentric ring-like focusing zones that can be seen as red zones in FIG. As the epitakophoresis zone moves from the edge of the device towards the center, eventually the SPADNS focusing zone enters the center of the device and is collected in the center of the device, thereby providing the desired epitakophoresis using epitakophoresis. Sample focusing and recovery was demonstrated.

Epitacophoresis was performed to separate and focus the SPADNS and Patent Blue dyes using acetic acid as a spacer using the epitakophoresis apparatus of Figures 9A-9B. 20 mM HCl-histidine (pH 6.20) was used as the pre-electrolyte. 5 mL of 10 mM MES Tris (pH 8.00) was used as trailing electrolyte, which was further mixed with 150 μl of 10 mm acetic acid, 150 μl of 0.1 mM Patent Blue dye, and 150 μl of 0.137 mM SPADNS. Effective mobility values (10 ⁻⁹ m ² /Vs) for SPADNS, acetic acid and Patent Blue dyes were 55, 42, 7 and 32, respectively. The electrode reservoir of the device contained the pre-electrolyte His (pH 6.25) at a concentration of 100 mM. No gel was used in the channels of this device for this experiment, but gel was present on top of the device platform.

Referring to FIG. 11, the device was filled with a mixture of trailing electrolyte, SPADN, acetic acid, and Patent Blue dye. A constant power of 2 W was used to perform epitachophoresis. SPADNS is focused into concentric ring-shaped focusing zones, visible as red zone/inner zone in FIG. 11, and Patent Blue dye is also focused into concentric ring-shaped focusing zones, visible as blue zone/outer zone in FIG. Focused. As the epitakophoresis zone moves from the edge of the device towards the center, eventually the focused zones of SPADNS and Patent Blue dyes sequentially enter the center of the device and can be collected separately at the center of the device, Separation, focusing and recovery of desired samples using epitakophoresis were thereby demonstrated.

Example 6: Device for epitachophoresis

An epitakophoresis device was designed to perform epitakophoresis (Fig. 12). The device of Figure 12 had a circular configuration and a circular copper tape electrode with a diameter of 5.8 cm.

Example 7: System for performing epitakophoresis using conductivity-based sample detection

Equipment construction

According to the previous examples, an epitacophoresis device with a large sample volume capacity was machined with circular separation channels. Figures 13A and 13B show two views of the structure of the device. A wiring electrode (1 mm diameter stainless steel wire; 55 mm radius) was attached to the edge of the circular separation compartment. The sample volume was defined by the space between the ring electrode and the agarose-stabilized leading electrolyte disc (35 mm radius). The applicable sample volume was therefore 5.7 mL per mm of its height. A second electrode was placed in the preceding electrolyte reservoir on the side of the device. The ring electrode was connected to the top banana connector shown in FIG. 13A. The bottom banana connector was attached to a 3 cm long, 0.4 mm diameter platinum wire electrode placed in the lead electrode reservoir (“b” in the scheme shown in FIG. 13B). An internal channel of 9 mm internal diameter with a total length of 20 cm was drilled into the interior of the device to prevent possible interference from electrolysis products migrating from the leading electrolyte reservoir towards the central collection well ("a" in the scheme shown in Figure 13B). . The post-drilling side opening of the device was blocked by a silicon septum. A central collection well of 9 mm diameter was drilled into the device and closed from the bottom by a movable Ertacetal® rod sealed with a rubber O-ring.

For all analyses, plastic vials with semi-permeable membranes (Slide-A-Lyzer™ MINI Dialysis Unit 2000 Da MWCO, Thermo Fisher Scientific, USA) were placed in the central collection well after filling with preceding electrolyte up to the lead electrode reservoir. inserted into the To minimize volume, the Slide-A-Lyser was cut in half with a razor blade to produce a collection cup with a volume of less than 200 microliters. Next, a 0.3% agarose gel disk (70 mm diameter, 4 mm thick) with a 8 mm center hole was prepared in the preceding electrolyte and placed in the center of the device, also 8 mm center to avoid air bubble accumulation. It was covered with a 75×1 mm circular glass plate with holes. Although various electrophoretic separation modes can be applied (e.g., zone electrophoresis, isoelectric focusing, or displacement electrophoresis), we include leading (LE) and terminating (TE) electrolytes. Epitacophoresis with an electrolyte system was used. The sample solution in the terminal electrolyte was applied by syringe into the space between the gel disk and the ring electrode. The polarity of the current connections was chosen such that the anionic sample components migrated from the ring electrodes towards the collection wells in the center of the device. After the focused sample zone entered the collection cup, the current was turned off and the sample was pipetted out for further use. The empty collection cup was lifted with the transfer bar and discarded.

Electric drive separation conditions

Separation was performed in negative mode with Cl ⁻ ions acting as precursor ions (effective mobility 79.1×10 ⁻⁹ m ² V ⁻¹ s ⁻¹ ). The leading electrolyte (LE) contained 100 mM HCl-histidine buffer pH 6.2 and the terminal electrolyte (TE) contained 10 mM TAPS titrated to pH 8.30 by TRIS. Agarose-stabilized preelectrolyte discs were prepared in 20 mm preelectrolyte (HCl-histidine; pH 6.25). All buffers were prepared in deionized water. The power supply was provided by a PowerPac 3000 (BioRad), which was run in constant power mode at 2W (which corresponds to approximately 16mA and 120V at the start of the analysis). The analysis took approximately 1 hour (approximately 10 mA and 200 V at the end of the analysis).

Sample detection

To detect the samples, a surface resistivity detection cell was constructed and connected to the conductivity detector of a commercial ITP instrument (Villa Labeco, Sp.N.Ves, Slovakia). A detection cell was prepared as follows: Two platinum (Pt) wires (300 μm×2 cm long) were attached to connectors compatible with the ITP instrument. Both ends of the Pt wire were inserted into a 1 mL pipette tip, which was then filled with a fast-curing epoxy resin. Finally, 1 mm of the pipette tip with the epoxy wire embedded inside was cut with a razor blade exposing a flat epoxy surface with two round Pt electrodes. See Figures 14A and 14B. The detection cell was mounted on a bench gently touching the surface of the agarose gel disk near the collection vial, as illustrated in Figure 15A. This detection system was used to generate the conductivity traces of Figures 15B and 17B.

Another exemplary system was also constructed for sample detection during epitachophoresis. In this system, a surface resistivity sensing probe consisting of two platinum (Pt) wires with a diameter of 500 μm is incorporated into the lower substrate (i.e., lower plate) of the epitachophoresis device, as shown in FIGS. 16A and 16B. is. The tip of the wire was close to the semipermeable membrane from below via a dedicated channel in the central post on the bottom substrate. Both ends of the wire were connected to the conductivity detector of a commercial ITP device (Villa Labeco, Sp.N.Ves, Slovakia). The top plate, which functions as an epitakophoresis device, is assembled with the bottom substrate using magnets, while an O-ring (see FIG. 16B) provides a perfect seal between the two substrates to prevent leakage. made it possible to stop

Chemical substance

Buffer component: L-histidine monohydrochloride monohydrate (99%), L-histidine (99%), N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (tape; 99.5%) and tris-(hydroxymethyl)aminomethane (Tris; 99.8%) were purchased from Sigma-Aldrich (USA). Agarose with low electropenetration NEEO ultra-quality Roti® garose was purchased from Carl Roth (Germany). Acetic acid and the anionic dye Patent blue V sodium salt were obtained from Sigma-Aldrich. The red anionic dye SPADNS (1,8-dihydroxy-2-(4-sulfophenylazo)naphthalene-3,6-disulfonic acid trisodium salt was obtained from Lachema, Brno, Czech Republic.

Focus of SPADNS and Patent Blue

To test the above exemplary devices including epitakophoresis and electrical sample detection, the devices were used to focus and detect test analytes: SPADNS and Patent Blue. Leading Electrolyte (LE): HCl-HIS buffered to pH 6.2. Trailing electrolyte (TE): TAPS-TRIS was buffered to pH 8.3. Gels were formed from 20 mL of polyacrylamide gel 6% in 20 mm LE. 100 mM LE was added to the electrode reservoir. Sample solution: 15 mL 10 mM TE + 150 μl 0.1 mM SPADNS + 150 μl 0.1 mM Patent Blue. The sample solution in the terminal electrolyte was applied by syringe into the space between the gel disk and the ring electrode. The device was run in constant power mode with P=2W. FIG. 15A provides images of SPADNS and Patent Blue focusing, showing conductivity sample detection near the sample collection well. FIG. 15B provides the conductivity trace for this sample focus, showing the significant change in conductivity/resistivity with the transition between LE and TE encompassing the sample focus zones (SPADNS and Patent Blue). there is

DNA analysis

Fluorescein-labeled low molecular weight dsDNA ladders (10 fragments of 75 bp-bp to 1622 bp) were from Bio-Rad, USA. DNA concentrations in collected fractions were assessed using a Qubit fluorometer (Invitrogen, Carlsbad, Calif., USA) by using a sensitive dsDNA Qubit quantitative assay kit. Concentrations of target molecules in samples were reported by fluorescent dyes that emit light only when bound to DNA. Collected fractions were further analyzed using a chip CGE-LIF instrument Agilent 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA). This analysis provided size information of the recovered DNA fragments in the samples using a highly sensitive DNA reagent kit (Agilent, USA).

DNA focusing

The electrophoretic mobility of DNA fragments greater than 50 bp is approximately 37×10 ⁻⁹ m ² /Vs in free solution, and short fragments (approximately 20-50 bp) can be shifted by only about 10%. Based on these mobilities, we designed a discontinuous electrolyte system suitable for focusing all sample DNA fragments into a single concentration zone. For experimental testing, we selected fluorescein-labeled small-range DNA ladders with fragment sizes ranging from 75 to 1632 bp. Fluorescence of samples with only one fluorophore per DNA fragment was illuminated by a 2 cm radius laser beam (Fig. 17A). Surface resistivity detection was used to show the transition of the LE/TE boundary close to the collection well (conductivity trace shown in Figure 17B). The overall change in resistivity from LE to TE was used as an indicator of sample position. Based on this change, the voltage was turned off and the separation stopped. Collected fractions were analyzed by both UV spectroscopy (absorbance measurements) (Figure 17C) and bioanalyzer-based analysis (Figure 17D). The lower signal intensity of the anterior and posterior markers in Figure 17D (added in equal amounts to initial and final samples according to manufacturer's instructions) was due to higher DNA concentration in the collected fractions. rice field. In both cases, an approximately 30-fold increase in concentration of the collected fractions corresponded to a decrease in sample volume from a starting 15 mL to a sample collection volume of 280 μl in this exemplary embodiment. The volume of the migrating DNA zone before entering the collection cup is much smaller (approximately 3 μl) and the final fraction concentration depends mainly on the volume of the collection vial chosen.

Example 8: ETP with voltage-based sample detection

In this example, three independent ETP runs were performed to focus and collect cfDNA from 1 mL of plasma using the ETP device, and the voltage was measured during the time course of each of the three independent ETP runs. bottom. Each of the three ETP runs was 75 minutes long. The power level at the start of each ETP run was 6W. The power level was then lowered to 3W for 30 minutes and then to 2W for 60 minutes. Results obtained during each of three independent ETP runs are shown in FIG.

Referring now to FIG. 18, in each stage the power was kept constant, the voltage gradually increased. In the final stage (2W output, 60-75 minutes), the voltage was 65V as the focusing zone containing nucleic acid molecules moved into the collection cup. Comparing each of three independent runs, the voltage profile was observed to be consistent, indicating that voltage feedback from the power supply can be used to monitor the position of nucleic acid molecules within the device.

Example 9: ETP with optical-based sample detection

In this example, the ETP run was performed using optical detection with a colored dye to monitor the position of the nucleic acids during the ETP run. Dyes with lower electrophoretic mobilities than nucleic acids can be used to allow optical tracking of the position of nucleic acids. For example, such dyes include Brilliant Blue FCF, Indigo Carmine, Sunset Yellow FCF, Allura Red, Fast Green FCF, Patent Blue V and Carmoisine. In this example, two independent ETP runs were performed to focus and collect nucleic acid molecules using brilliant blue dye as an optical marker (ETP Run 1: FIGS. 19A-19B; and ETP Run 2 : see FIGS. 19C-19D).

FIG. 19A shows an image of an ETP device during an ETP run using Brilliant Blue dye as an optical marker during focusing and collection of nucleic acid molecules, and SYBR-Gold dye in addition to monitor the position of the nucleic acid molecules. The electrophoretic mobility of the blue dye is lower than that of the nucleic acid, so the blue dye migrated after the focused zone containing the nucleic acid. Contaminants appeared as brown focused zones (see Figure 19A). In addition to the photographic images in Figure 19A, fluorescence-based images were taken during the ETP run (see Figure 19B). The fluorescence-based image in FIG. 19B demonstrates that the focal zone containing bands containing DNA labeled with SYBR-Gold migrated faster than the brilliant blue dye.

FIG. 19C shows an image of the ETP device during ETP using Brilliant Blue as an optical marker during focusing and collection of nucleic acid molecules. Plasma samples containing cfDNA were used for the ETP runs of Figures 19C and 19D. The ETP run was stopped when the dye band reached the collection wells. After focusing and collecting the cfDNA, analysis was performed on the focused and collected cfDNA samples using the Agilent TapeStation system (see Figure 19D). The electropherogram (see Figure 19D) shows a 179 bp peak representing the desired cfDNA molecule focused and collected during the ETP run, thereby demonstrating the usefulness of the Brilliant Blue dye for monitoring the location of nucleic acids. demonstrated the sex.

Example 10: ETP with heat-based sample detection

In this example, an ETP run using thermal imaging during DNA ladder focusing and collection was performed. An infrared-based thermal imaging camera (SEEK thermal ShotPro) was used for thermal imaging in this example. Additionally, fluorescence imaging was used. Thermal and fluorescent images were taken at four consecutive time points of 20, 40, 42, and 44 minutes. (See FIGS. 20A-20B). Additionally, voltage feedback from the power supply was measured during the ETP run.

FIG. 20A shows thermal images taken at four time points during ETP: 20, 40, 42, and 44 minutes. We observed that the temperature in the center of the device increased by 17° C. (38° C. to 55° C.) between 40 and 44 minutes, during which time the DNA ladder visible as green fluorescent circles in FIG. 20B migrated into the central collection cup. was done.

FIG. 20C shows voltage and temperature changes over time during the ETP run of this example. The trend of voltage change over time was observed to be similar to the trend of temperature change over time. Voltage feedback from the power supply thus provided an additional means to monitor the position of the DNA ladder molecules within the device.

Example 11: Isolation/purification of total nucleic acids by ETP

Nucleic acids, including DNA and RNA, from samples, including formalin-fixed paraffin-embedded tissue (“FFPET”) samples, to perform ETP using an epitacophoresis apparatus and experimental setup (see FIGS. 21A-21B) was isolated (purified).

Prior to setting up and running the ETP to isolate/purify nucleic acids from the FFPET samples, the ETP buffer, the agarose gel of the ETP device, and the shortened dialysis unit were prepared. A leading electrolyte (“LE”) buffer containing HCl-histidine pH 6.25 was prepared and a trailing electrolyte (“TE”) buffer containing TAPS-Tris pH 8.30 was prepared. Agarose gels used with the ETP device were prepared by mixing the appropriate amount of agarose for the desired agarose percentage gel with LE buffer in an Erlenmeyer flask.

FFPET samples were also prepared by deparaffinization and lysis of FFPET curls prior to setting up and performing ETPs to isolate/purify nucleic acids from FFPET samples. As discussed further in the Examples below, in some instances deparaffinization and dissolution of FFPET curls was performed using a Promega-based protocol, and in some instances deparaffinization and dissolution of FFPET curls. Lysis was performed using a KAPA Express Extract-based protocol.

ETP-based isolation/purification of nucleic acids from FFPET samples generally proceeded as follows. First, the ETP device was prepared for sample loading.
An FFPET sample solution was prepared, typically comprising 7 mL of TE buffer and approximately 110-220 μl of the dissolved FFPET sample prepared above, and pipetted into the gap between the gel and the circular electrode (FIGS. 21A-21D). 21B). When brilliant blue dye was used as a marker, 30 μl of brilliant blue solution was added.

The power supply was then primed by plugging the ETP device into the power supply. ETP was performed for approximately 30-40 minutes by setting the power supply to a constant power of 2 W and turning it on.

In some instances, samples were monitored and collected as follows. In the example using Brilliant Blue dye, white light (no filter) was used to monitor dye movement (see Figures 22A-22B). The ETP run was stopped when the dye was completely in the dialysis cup. The TE buffer and gel were removed and the ETP isolation/purification sample was collected from the dialysis unit.

In some instances, a cleanup step was performed on the isolated/purified samples. In these examples, whole isolated/purified ETP samples were added to Amicon centrifugal filters, such as Amicon centrifugal filters with 3 kDa or 10 kDa cut-off filters, and centrifugation-based clarification was performed. After centrifugation-based cleanup, concentrated nucleic acid samples were collected and optionally stored at −80° C. before future use.

In some instances, analysis of collected nucleic acids, such as DNA and/or RNA, was performed using a Qubit fluorometer (Invitrogen, Carlsbad, Calif., USA), which reported the concentration of nucleic acids. In some instances, analysis of collected nucleic acids, such as DNA and/or RNA, was performed using an Agilent TapeStation system (Agilent, Santa Clara, Calif., USA). This analysis provided size information about the collected nucleic acid fragments. In some instances, analysis of collected nucleic acids, such as DNA and/or RNA, was performed using qPCR-based analysis that yielded a Q-score that represents the quality of the nucleic acid. Note that Q-scores range from 0 (low quality) to 1 (high quality).

Example 12: Total Nucleic Acid Isolation/Purification from FFPET Samples by ETP, dsDNA Yield Analysis, and RNA Yield Analysis

In this example, ETP-based isolation/purification was used to extract total nucleic acid content FFPET samples from four different CRC blocks (CRC block #1 to CRC block #4) and two samples of healthy adjacent tissue. Isolate/purify and measure the yield of dsDNA and RNA from the ETP-based isolation and purification using only the Promega column-based method as generally described in Example 8. A comparison was made with the dsDNA and RNA obtained. dsDNA yield and RNA yield were quantified using a Qubit fluorometer. Two to four replicates of each run were performed and statistically significant results were those with P<0.03.

Referring now to Figure 23, ETP-based isolation/purification of dsDNA from CRC blocks 1-4 and healthy adjacent tissue yielded the desired levels of dsDNA in each case. In addition, ETP-based isolation/purification of dsDNA is superior to the Promega column-based method when nucleic acid input is low, e.g., in CRC block 3, CRC block 4, healthy adjacent tissue #1 and healthy adjacent tissue #2. A significant overrun was observed. Furthermore, it is noted that CRC block 4 and healthy adjacent tissue 2 and CRC block 1 results represented statistically significant results for P<0.03.

Referring now to Figure 24, ETP-based isolation/purification of RNA from CRC blocks 1-4 and healthy adjacent tissue samples yielded the desired levels of RNA in each case. Furthermore, it was observed that ETP-based isolation/purification of RNA yielded comparable or increased amounts of RNA compared to the Promega-based column method (see Figure 24).

Example 13: Analysis of Nucleic Acids Obtained by ETP-Based Isolation/Purification of FFPET Samples

In this example, nucleic acids obtained by ETP-based isolation/purification were analyzed by Agilent TapeStation-based analysis to examine the size profile of the isolated/purified dsDNA. In addition, dsDNA obtained by ETP-based isolation/purification was analyzed by quality control qPCR to obtain a Q-score (a measure of nucleic acid quality as described above). Four ETP-based isolation/purification runs were performed to generate the data in Figure 25A and these data were compared to four controls. In addition, 4 ETP-based runs with samples from CRC blocks 1, 2, 3 and healthy adjacent tissue #2 were performed using the same 4 sample sources: CRC blocks 1, 2, 3 and healthy dsDNA Q-scores obtained using the Promega column-based method using adjacent tissue #2 (Fig. 25B).

Referring now to FIG. 25A, the dsDNA obtained by ETP-based isolation/purification had a major peak at a size of approximately 900 bp, so a series of DNA sizes containing relatively large dsDNA was collected and further A peak was observed to be approximately 7000 bp. The results shown in Figure 25A demonstrated that the size profile of dsDNA obtained by ETP-based isolation/purification was similar to that of the control.

Referring now to Figure 25B, dsDNA obtained by ETP-based isolation/purification was observed to have similar or improved Q-scores compared to dsDNA obtained by the Promega column-based method, It demonstrated that high quality dsDNA was obtained using ETP-based isolation/purification.

Example 14: Total Nucleic Acid Isolation/Purification from FFPET Samples by ETP and Nucleic Acid Yield Analysis

In this example, ETP-based isolation/purification was used to isolate/purify total nucleic acid content FFPET samples from two different CRC blocks, and dsDNA and RNA from the ETP-based isolation and purification. Yields were compared to dsDNA and RNA obtained using the KAPA bead-based method as generally described in Example 8. dsDNA yield and RNA yield were quantified using a Qubit fluorometer. Two replicates were performed for each run and statistically significant results were those with P<0.05. Additionally, note that the KAPA Express Extract-based protocol, rather than the Promega-based protocol, was used prior to the ETP run for FFPET sample preparation.

Referring now to Figure 26A, ETP-based isolation/purification of dsDNA from CRC block 2 and CRC block 4 yielded the desired levels of dsDNA in each case. Furthermore, ETP-based isolation/purification of dsDNA resulted in higher yields of dsDNA from CRC block 2 compared to the KAPA bead-based method.

Referring now to Figure 26B, ETP-based isolation/purification of RNA from CRC block 2 and CRC block 4 yielded the desired levels of RNA in each case. Furthermore, ETP-based isolation/purification of dsDNA resulted in higher yields of RNA from CRC block 2 compared to the KAPA bead-based method.

Example 15: Total Nucleic Acid Isolation/Purification from FFPET Samples by ETP and Nucleic Acid Analysis

In this example, ETP-based isolation/purification was used to isolate/purify nucleic acids from FFPET samples, and the collected nucleic acids were subjected to DNase I treatment. As shown in Figure 27, two different DNase I treatments were performed on separate ETP isolation/purification samples: Sigma DNase I and NEB DNase I + column cleanup and collected after ETP-based isolation/purification. Four different nucleic acid samples were compared to four control samples both before and after DNase treatment of ETP-based samples and control samples by Agilent TapeStation-based analysis (see Figure 28).

Referring now to Figure 27, both DNase I treatments showed degradation of the DNA contained in each of the samples, thereby validating the RNA results of the previous example above.

Referring now to Figure 28, the isolated/purified nucleic acid samples (both control and ETP-based) exhibited size profile changes before and after DNaseI treatment consistent with DNaseI treatment, effectively purifying the DNA contained in the samples. This further confirmed the RNA results of previous examples above.

Example 16: Simultaneous extraction of DNA and RNA from FFPET (colorectal cancer)

In this example, we used the ETP described herein and for comparison a Promega FFPET DNA column cleanup (following the manufacturer's instructions, except omitting the RNase step). , extracted DNA and RNA from five CRC FFPET blocks. Nucleic acid yield was assessed using a Qubit fluorometer (ThermoFisher Scientific, Carlsbad, Calif.). DNA content was measured after RNase treatment and DNA column cleanup. RNA quantity was measured after DNase treatment and RNA column cleanup. Nucleic acid quality was assessed for ETP isolated material and DNA by PCR only. Quality is represented by a Q-score (range 0-1). The results are shown in FIG. The results demonstrate that the methods and devices disclosed herein exceed the performance of state-of-the-art methods in the 8/10 test.

For each extraction method, two extraction replicas were subjected to sequencing on the Illumina platform. Two sequencing runs were performed for each replica (4 sequencing runs for each extraction method). Sequencing libraries were prepared and target enrichment was performed using the AVENIO Library Prep kit (Roche Sequencing Solutions, Pleasanton, Calif.). Sequencing results are shown in FIG. Sequencing data obtained using nucleic acids isolated as disclosed herein are excellent in nearly every metric tested.

Various steps are described in the above procedure. It will, however, be evident that various modifications and changes may be made, and additional procedures may be performed, without departing from the broader scope of the procedures set forth in the appended claims.

Claims (13)

A method of isolating nucleic acids from a formalin-fixed, paraffin-embedded tissue (“FFPET”) sample, said method comprising:
i. providing a device for performing epitachophoresis (ETP) comprising a gel containing a leading electrolyte (LE), a central electrode, and a circular outer electrode surrounding a collection well;
ii. preparing an FFPET sample solution in a trailing electrolyte (TE) from the FFPET sample;
iii. performing epitachophoresis by applying a constant power to said device to focus said nucleic acid into one or more focusing zones; and iv. collecting the one or more focus zones from the collection well, thereby obtaining a solution of isolated nucleic acids. 2. The method of claim 1, wherein the FFPET sample preparation comprises deparaffinization and lysis. 2. The method of claim 1, wherein the FFPET sample solution further comprises a dye, and the method further comprises monitoring migration of the dye and collecting the focusing zone when the dye reaches the collection well. described method. 2. The method of claim 1, wherein said nucleic acid is a combination of DNA and RNA. 2. The method of claim 1, wherein said isolated nucleic acid is of a predetermined size.
i. 5. 2. The method of claim 1, further comprising isolating DNA by treating the solution of isolated nucleic acids with RNase. 2. The method of claim 1, further comprising isolating RNA by treating the solution of isolated nucleic acids with DNase. 2. The method of claim 1, wherein the preceding electrolyte comprises HCL-histidine. 2. The method of claim 1, wherein the trailing electrolyte comprises MES or TAPS. A system for isolating nucleic acids from formalin-fixed, paraffin-embedded tissue (“FFPET”) samples, said system comprising:
i. an apparatus for performing epitachophoresis (ETP), comprising a circular outer electrode surrounding a gel containing a leading electrolyte (LE), a sample loading area, a central electrode, and a collection well;
ii. a power supply;
iii. and means for collecting samples from said collection wells. 11. The system of claim 10, further comprising means for monitoring migration of said nucleic acids through said gel. 11. The system of claim 10, further comprising means for coupling said monitoring to said collection. 11. The system of claim 10, further comprising means for loading a sample into said sample loading area.

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